Toxoplasma gondiiis a cosmopolitan intracellular protozoan whose success as a pathogen is attributable to the creation of a nonfusogenic parasitophorous vacuole (PV). Endocytosed within the PV, parasites replicate in a niche sequestered from host cell endocytic and exocytic traffic. This unique compartment is established during invasion, which effects the intimate apposition of host cell and parasite plasma membranes. The intercellular interface, the "moving junction," is likely the primary regulator of PV membrane (PVM) composition and PVM nonfusogenicity, hence, parasite survival. Remarkably, since its discovery decades ago, this membranous contact region between host and parasite has remained largely uncharacterized with regard to precise function, macromolecular organization, and biochemical content. This proposal will test the hypothesis that the moving junction is a biochemically-defined entity that governs the initial attributes of the protective vacuolar membrane.The first specific aim will demonstrate that the moving junction selectively directs the entry of cholesterol and charged phospholipids into the PVM and sorts host cell transmembrane proteins by virtue of membrane microdomain association. The second specific aim will test whether the moving junction is itself comprised of lipid rafts. Finally, the third specific aim will utilize an elegantly designed strategy to identify host cell and parasite moving junction proteins. Progress in these endeavors promises to augment our appreciation of the complexity of this specialized heterotypic junction, and to leave us poised to utilize this knowledge to combat a particularly pernicious phylum of intracellular human pathogens.